Fig. 5. Effect of the chronic presence of insulin and gliclazide on extracellular-signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 activation. C2C12 cells, differentiated in the absence (MF) or in the chronic presence (MFI) of insulin, were washed with Krebs-Ringer phosphate (KRP) buffer as described in Materials and methods, followed by stimulation with insulin (100 nmol l^-1) for 5 min. Lysates were prepared from cells treated with 2 mmol l^-1 gliclazide during the last 24 h of differentiation. Protein was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with phospho-specific ERK (pERK) (A), phospho-specific JNK (pJNK) (D), phospho-specific p38 (pp38) (G), ERK (B), JNK (E), p38 (H) antibodies. Phospho-specific blots are representative of experiments performed three times. Error bars of quantified data for pERK (C), pJNK (F) and pp38 (I) represent the S.E.M. of three independent experiments.